Isolated decapeptides which bind to HLA molecules

ABSTRACT

Nonapeptides and decapeptides which bind to HLA molecules and provoke proliferation of cytolytic T cells are disclosed. The decapeptides terminate in Valine, and are restricted in their first three amino acid positions. Other useful nonapeptides are also disclosed.

This application is a divisional Ser. No. 08/880,963, filed Jun. 23,1997, now U.S. Pat. No. 6,025,470, incorporated by reference.

FIELD OF THE INVENTION

This invention relates to peptides which are useful in the context ofcellular immunology. More particularly, the invention relates topeptides which bind to HLA molecules on the surface of cells. At leastsome of these peptides also induce the activation of cytolytic T cells,when they are complexed with their partner HLA molecule. Also a part ofthe invention are the uses of these peptides in areas such asidentifying HLA-A2 positive cells, provoking T cells, determiningpresence of particular T cells, as well as cytolytic T cells themselves.

BACKGROUND AND PRIOR ART

The study of the recognition or lack of recognition of cancer cells by ahost organism has proceeded in many different directions. Understandingof the field presumes some understanding of both basic immunology andoncology.

Early research on mouse tumors revealed that these displayed moleculeswhich led to rejection of tumor cells when transplanted into syngeneicanimals. These molecules are “recognized” by T-cells in the recipientanimal, and provoke a cytolytic T-cell response with lysis of thetransplanted cells. This evidence was first obtained with tumors inducedin vitro by chemical carcinogens, such as methylcholanthrene. Theantigens expressed by the tumors and which elicited the T-cell responsewere found to be different for each tumor. See Prehn, et al., J. Natl.Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572(1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30:2458-2462 (1970) for general teachings on inducing tumors with chemicalcarcinogens and differences in cell surface antigens. This class ofantigens has come to be known as “tumor specific transplantationantigens” or “TSTAs”. Following the observation of the presentation ofsuch antigens when induced by chemical carcinogens, similar results wereobtained when tumors were induced in vitro via ultraviolet radiation.See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types oftumor described supra, spontaneous tumors were thought to be generallynon-immunogenic. These were therefore believed not to present antigenswhich provoked a response to the tumor in the tumor carrying subject.See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

The family of tum⁻ antigen presenting cell lines are immunogenicvariants obtained by mutagenesis of mouse tumor cells or cell lines, asdescribed by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), thedisclosure of which is incorporated by reference. To elaborate, tum⁻antigens are obtained by mutating tumor cells which do not generate animmune response in syngeneic mice and will form tumors (i.e., “tum⁺”cells). When these tum⁺ cells are mutagenized, they are rejected bysyngeneic mice, and fail to form tumors (thus “tum⁻”). See Boon et al.,Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which isincorporated by reference. Many tumor types have been shown to exhibitthis phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

It appears that tum⁻ variants fail to form progressive tumors becausethey initiate an immune rejection process. The evidence in favor of thishypothesis includes the ability of “tum⁻” variants of tumors, i.e.,those which do not normally form tumors, to do so in mice with immunesystems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl.Acad. Sci. USA 76: 5282-5285 (1979); and the observation thatintraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called “tumor rejection antigen”in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearon et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

A class of antigens has been recognized which are presented on thesurface of tumor cells and are recognized by cytolytic T cells, leadingto lysis. This class of antigens will be referred to as “tumor rejectionantigens” or “TRAs” hereafter. TRAs may or may not elicit antibodyresponses. The extent to which these antigens have been studied, hasbeen via cytolytic T cell characterization studies, in vitro i.e., thestudy of the identification of the antigen by a particular cytolytic Tcell (“CTL” hereafter) subset. The subset proliferates upon recognitionof the presented tumor rejection antigen, and the cells presenting thetumor rejection antigens are lysed. Characterization studies haveidentified CTL clones which specifically lyse cells expressing the tumorrejection antigens. Examples of this work may be found in Levy et al.,Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152:1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980);Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski etal., Eur. J. Immnunol. 12: 406-412 (1982); Palladino et al., Canc. Res.47: 5074-5079 (1987). This type of analysis is required for other typesof antigens recognized by CTLs, including minor histocompatibilityantigens, the male specific H-Y antigens, and the class of antigensreferred to as “tum−” antigens, and discussed herein.

A tumor exemplary of the subject matter described supra is known asP815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens—and this is a key distinction—the tum⁻ antigensare only present after the tumor cells are mutagenized. Tumor rejectionantigens are present on cells of a given tumor without mutagenesis.Hence, with reference to the literature, a cell line can be tum⁺, suchas the line referred to as “P1”, and can be provoked to produce tum⁻variants. Since the tum⁻ phenotype differs from that of the parent cellline, one expects a difference in the DNA of tum⁻ cell lines as comparedto their tum⁺ parental lines, and this difference can be exploited tolocate the gene of interest in tum⁻ cells. As a result, it was foundthat genes of tum variants such as P91A, 35B and P198 differ from theirnormal alleles by point mutations in the coding regions of the gene. SeeSzikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989).This has proved not to be the case with the TRAs of this invention.These papers also demonstrated that peptides derived from the tum⁻antigen are presented by H-2^(d) Class I molecules for recognition byCTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

PCT application PCT/US92/04354, filed on May 22, 1992 assigned to thesame assignee as the subject application, teaches a family of humantumor rejection antigen precursor coding genes, referred to as the MAGEfamily. Several of these genes are also discussed in van der Bruggen etal., Science 254:1643 (1991). It is now clear that the various genes ofthe MAGE family are expressed in tumor cells, and can serve as markersfor the diagnosis of such tumors, as well as for other purposesdiscussed therein. See also Traversari et al., Immunogenetics 35: 145(1992); van der Bruggen et al., Science 254: 1643 (1991) and De Plaen,et al., Immunogenetics 40: 360 (1994). The mechanism by which a proteinis processed and presented on a cell surface has now been fairly welldocumented. A cursory review of the development of the field may befound in Barinaga, “Getting Some ‘Backbone’: How MHC Binds Peptides”,Science 257: 880 (1992); also, see Fremont et al., Science 257: 919(1992); Matsumura et al., Science 257: 927 (1992); Engelhard, Ann. Rev.Immunol 12:181-207 (1994); Madden, et al, Cell 75:693-708 (1993);Ramensee, et al, Ann. Rev. Immunol 11:213-244 (1993); German, Cell 76:287-299 (1994). These papers generally point to a requirement that thepeptide which binds to an MHC/HLA molecule be nine amino acids long (a“nonapeptide”), and to the importance of the second and ninth residuesof the nonapeptide. For H-2K^(b), the anchor residues are positions 5and 8 of an octamer, for H-2D^(b), they are positions 5 and 9 of anonapeptide while the anchor residues for HLA-A1 are positions 3 and 9.of a nonamer. Generally, for HLA molecules, positions 2 and 9 areanchors.

Studies on the MAGE family of genes have now revealed that a particularnonapeptide is in fact presented on the surface of some tumor cells, andthat the presentation of the nonapeptide requires that the presentingmolecule be HLA-A1. Complexes of the MAGE-1 tumor rejection antigen (the“TRA” or nonapeptide”) leads to lysis of the cell presenting it bycytolytic T cells (“CTLs”).

Research presented in, e.g., U.S. Pat. No. 5,405,940 filed Aug. 31,1992, and in U.S. Pat. No. 5,571,711, found that when comparinghomologous regions of various MAGE genes to the region of the MAGE-1gene coding for the relevant nonapeptide, there is a great deal ofhomology. Indeed, these observations lead to one of the aspects of theinvention disclosed and claimed therein, which is a family ofnonapeptides all of which have the same N-terminal and C-terminal aminoacids. These nonapeptides were described as being useful for variouspurposes which includes their use as immunogens, either alone or coupledto carrier peptides. Nonapeptides are of sufficient size to constitutean antigenic epitope, and the antibodies generated thereto weredescribed as being useful for identifying the nonapeptide, either as itexists alone, or as part of a larger polypeptide.

The preceding survey of the relevant literature shows that variouspeptides, usually eight, nine, or ten amino acids in length, complexwith MHC molecules and present targets for recognition by cytolytic Tcells. A great deal of study has been carried out on melanoma, andmelanoma antigens which are recognized by cytolytic T cells are nowdivided into three broad categories. The first, which includes many ofthe antigens discussed, supra, (e.g., MAGE), are expressed in somemelanomas, as well as other tumor types, and normal testis and placenta.The antigens are the expression product of normal genes which areusually silent in normal tissues.

A second family of melanoma antigens includes antigens which are derivedfrom mutant forms of normal proteins. Examples of this family are MUM-1(Coulie, et al, Proc. Natl. Acad. Sci. USA 92:7976-7980 (1995)); CDK4(Wölfel, et al, Science 269:1281-1284(1955)); Bcatenin (Robbins, et al,J. Exp. Med. 183:1185-1192 (1996)); and HLA-2 (Brandel, et al, J. Exp.Med. 183:2501-2508 (1996)). A third category, also discussed, supra,includes the differentiation antigens which are expressed by bothmelanoma and melanocytes. Exemplary are tyrosinase gp100, gp75, andMelan A/Mart-1. See U.S. Pat. No. 5,620,886 incorporated by reference,with respect to Melan-A. See Wölfel, et al., Eur. J. Immunol. 24: 759(1994) and Brichard, et al., Eur. J. Immunol. 26: 224 (1996) fortyrosinase; Kang, et al., J. Immunol. 155: 1343 (1995); Cox, et al.,Science 264: 716 (1994); Kawakami, et al., J. Immunol. 154: 3961 (1995)for gp 100; Wang, et al., J. Exp. Med. 183: 1131 (1996) for gp 75.

Cytolytic T cells (“CTLs” hereafter) have been identified in peripheralblood lymphocytes, and tumor infiltrating lymphocytes, of melanomapatients who are HLA-A*0201 positive. See Kawakami, et al, Proc. Natl.Acad. Sci. USA 91:3515 (1994); Coulie, et al, J. Exp. Med. 180:35(1994). When ten HLA-A*0201 restricted Melan-A specific CTLs werederived from different patients were tested, nine of them were found torecognize and react and the peptide Ala Ala Gly Ile Gly lie Leu Thr Val,(SEQ ID NO:2), which consists of amino acids 27-35 of Melan-A.(Kawakami, et al, J. Exp. Med 180:347-352 (1994)). Rivoltini, et al, J.Immunol 154:2257 (1995), showed that Melan-A specific CTLs could beinduced by stimulating PBLs from HLA-A*0201 positive normal donors, andmelanoma patients, using SEQ ID NO:2. The strength of this response hasled to SEQ ID NO:2 being proposed as a target for vaccine development.It has now been found, however, that a decapeptide, i.e.,

Glu Ala Ala Gly Ile Gly Ile Leu Thr Val

(SEQ ID NO:1), is actually a better target than SEQ ID NO:2. Thisrecognition has led to work set forth herein, which is part of theinvention.

The majority of peptides which have been identified as binding toHLA-A*0201 are 9 or 10 amino acids in length, and are characterized bytwo anchor residues. The first is Leu or Met at position 2, and thesecond is Leu or Val at position 9. See Falk, et al, Nature 351:290(1991). Ruppert, et al, in Cell 74:929 (1993), show that amino acidsfound at other positions within a nona- or decapeptide may also have arole in the peptide -HLA-A*0201 interaction. They show, e.g., that anegatively charged residue or proline at position 1 was associated withpoor HLA-A*0201 binding.

What is interesting about this work is that the two peptides representedby SEQ ID NOS:1 and 2 do not possess the major anchor residue atposition 2 and, the strong binder SEQ ID NO:1 has a negative residue atposition 1.

A strong binder is not necessarily a stable binder, meaning that theinteraction between peptide and HLA molecule may be, and is, brief. Whenit is desired to induce CTLs, to identify them or to carry out othertypes of experiments, it would be desirable to have a peptide with theability to bind to an MHC Class I molecule which binds with highaffinity and forms stable complexes.

The invention involves, inter alia, the development of new nonamers anddecamers which are surprisingly good HLA binders and CTL stimulators.These molecules, as well as their uses, are among the features of theinvention which are set forth in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B i.e. show results of experiments carried out todetermine if tumor infiltrating lymphocyte populations would lyse cellspresenting complexes of HLA-A*0201 and various peptides on theirsurfaces.

FIGS. 2A-2D show stability studies comparing various peptides.

FIG. 3 shows antigenic activity of various peptides when tested withTILs.

FIG. 4 parallels FIG. 3, but uses CTLs generated from PBLs bystimulation with various peptides i.e. SEQ ID NOS: 2, 1, and 9.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

In these experiments tumor infiltrating lymphocytes (“TILs” hereafter),were generated from tumor invaded lymph nodes of patients who wereHLA-A*0201 positive. The experiments were designed so as to avoidantigen specific selection in vitro, and the methodology is now setforth.

Biopsies of tumor infiltrated lymph nodes (“TILNs” hereafter) were lysedto single cell suspensions, and then cultured in 24 well culture plates.A total of 50 μl of cell suspension was added to 2 mls of Iscove'sDulbecco medium which had been supplemented with Asn (0.24 mM), Arg(0.55 mM), and Gln (1.5 mM), and 10% pooled human A⁺ serum (serumobtained from type A blood donors), together with recombinant human IL-2(100 u/ml), and IL-7 (10 ng/ml). These were the only cytokines used toculture the cell suspensions, so as to avoid antigen specific selectionin vitro. The suspensions were cultured for 2-3 weeks, and the cellswere then characterized for cell surface phenotype. Only populationswith greater than 75% CD8⁺T cells, and of desired cytolytic activitywere used. This second property was determined by combining the TILNpopulations with autologous cells, a melanoma cell line previouslycharacterized as being HLA-A*0201 positive (Me290), a melanoma cell lineknown to be HLA-A*0201 negative (Me260) or cell line T2, which does notprocess antigen, together with the peptide of SEQ ID NO:1. The peptidewas added at 1 μM, together with varying ratios of effector (TILN)cells, and the target cells. The results presented in FIG. 1, showresults obtained using LAU 132 and LAU 203, two TILN populationsidentified via this method. In FIG. 1, “M10” is SEQ ID NO:1, and theadditional abbreviations are as set forth, supra. The assay was a 4 hour⁵¹Cr release assay, carried out in the absence or presence ofexogenously added peptide. In FIG. 1, open symbols stand for the absenceof the peptide, and solid symbols for its presence. In this assay, thetarget cells were labelled with ⁵¹Cr for one hour, at 37° C., and werethen washed two times. Labelled cells (1000 cells in 50 μl) were addedto a 50 μl sample of effector cells (varying amounts, as indicatedherein), in the presence or absence of 50 μl of antigenic peptide (1μg/ml). Prior to their addition, the effector cells had been incubatedfor a minimum of 20 minutes at 37° C., in the presence of unlabellednatural killer (NK) cells (50,000 cells per well), in order to eliminateany non-specific lysis due to NK-like effectors present in the effectorpopulation. The ⁵¹Cr release was measured after 4 hours of incubation at37° C., and percent specific lysis was calculated as:$100\lbrack \frac{( {{{experimental}\quad {release}} - {{spontaneous}\quad {release}}} )}{{total} - {{spontaneous}\quad {release}}} \rbrack$

As FIG. 1 shows, the two TILN populations lysed the HLA-A*0201 positivecell line equally well, whether or not the peptide was added. TheHLA-A*0201 negative line, Me260, was not lysed in either situation, andT2, which does not process antigen, was lysed only when the peptide wasadded. These results show that the two TILN populations used hereafterrecognize the epitope defined by SEQ ID NO:1, when complexed toHLA-A*0201 positive cells.

EXAMPLE 2

The experiments described, supra, were modified somewhat, to determineif the TILNs recognized other peptides better than SEQ ID NO:1. In theseexperiments, the following peptides were synthesized, using knownmethods:

Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:2)

Ala Ala Gly Ile Gly Ile Leu Thr Val Ile (SEQ ID NO:3)

Ile Leu Thr Val Ile Leu Gly Val Leu (SEQ ID NO:4)

These peptides correspond, respectively, to amino acids 27-35, 27-36,and 32-40 of Melan-A.

TIL recognition was determined by incubating T2 cells (target), withTILs (effector), at an effector: target ratio of 30:1. Varyingconcentrations of the peptides SEQ ID NOS:1, 2, 3 or 4 were used. The⁵¹Cr release assay discussed, supra, was used. The following Table setsforth the results of these experiments, wherein the peptideconcentration is that which gave 50% of maximum activity. Relativeactivity is that obtained via comparison to SEQ ID NO:2, i.e.:[nM]50%[SEQ ID NO:2][nM]50%][test peptide].

TABLE I TILN LAU 203 TILN LAU 132 Peptide^(a) Peptide [nM] Relative [nM]Relative Peptide Sequence 50% activity^(b) 50% activity AAGIGILTV₂₇₋₃₅SEQ ID NO:2 40 1 15 1 EAAGIGILTV₂₆₋₃₅ SEQ ID NO:1 1.5 27 1 15AAGIGILTVI₂₇₋₃₆ SEQ ID NO:3 600 0.06 300 0.05 ILTVILGV₃₂₋₄₀ SEQ ID NO:4>10⁴ <4 × >10⁴ <1.5 × 10⁻³ 10⁻³

It will be seen that SEQ ID NO:1 had significantly higher activity thanthe other peptides tested.

EXAMPLE 3

A series of peptides were then synthesized, in order to attempt todetermine peptides with enhanced binding to HLA-A*0201 molecules. Thepeptides synthesized are considered to be derivatives of SEQ ID NO:2(i.e., Ala Ala Gly Ile Gly Ile Leu Thr Val), and are

Ala Leu Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:5)

Ala Met Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:6)

Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:7)

and

Met Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:8)

For SEQ ID NO:1, i.e., Glu Ala Ala Gly Ile Gly Ile Leu Thr Val thederivatives were:

Glu Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:9)

Glu Met Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:10)

Glu Ala Leu Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:11)

Glu Ala Met Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:12)

Tyr Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:13)

Phe Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:14)

Ala Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:15)

and

Ala Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO:16)

Three other control peptides were used, i.e.:

Glu Ile Leu Gly Phe Val Phe Thr Leu (SEQ ID NO:17)

Gly Val Asp Pro Ile Gly His Leu Tyr (SEQ ID NO:18) and

Phe Leu Trp Gly Pro Arg Ala Leu Val (SEQ ID NO: 19).

SEQ ID NOS: 17 and 18 correspond to amino acids 58-66 of Influenza Amatrix protein (“FLUMA”), and amino acids 168-176 of MAGE-3 TRAP.

In these experiments a peptide known to bind to HLA-A*0201 was used,i.e. amino acids 271-279 of the MAGE-3 TRAP (SEQ ID NO:20), in aninhibition assay, together with cytolytic T cell line 198NS (Valmori, etal., Canc. Res. 57:735 (1997) which recognizes complexes of SEQ ID NO:20and HLA-A*0201. In these assays, varying concentrations of test peptides(1 μM to 100 μM) were incubated with ⁵¹Cr labelled T2 cells (1000cells/well), for 15 minutes, at room temperature. A suboptimal dose ofSEQ ID NO:19 was then added (1 nM), together with CTL 198NS, in anamount sufficient to create a 5/1 effector/target ratio. A ⁵¹Cr releaseassay was then carried out, in accordance with the method set forth,supra. The amount of test peptide needed to inhibit recognition ofcomplexes by the CTL was calculated, and then binding affinity of eachpeptide, relative to SEQ ID NO:2, was calculated, using the formula:$R = \frac{{ID}_{50}( {{SEQ}\quad {ID}\quad {NO}\text{:}2} )}{{ID}_{50}( {{test}\quad {peptide}} )}$

If R is greater than one, then the tested peptide bound to HLA-A*0201with greater affinity than SEQ ID NO:2. A value less than one indicateslower affinity. The results are set forth below:

TABLE II Com- petitor^(a) Relative [μM] competitor Peptide Sequence 50%activity^(b) Melan-A₂₇₋₃₅ AAGIGILTV SEQ ID NO:2 60 1 ALGIGILTV SEQ IDNO:5 1.5 40 AMGIGILTV SEQ ID NO:6 2 30 LAGIGILTV SEQ ID NO:7 65 1MAGIGILTV SEQ ID NO:8 55 1 MeLan-A₂₆₋₃₅ EAAGIGILTV SEQ ID NO:1 5 4ELAGIGILTV SEQ ID NO:9 6.5 9 EMAGIGILTV SEQ ID NO:10 20 3 EALGIGILTV SEQID NO:11 100 0.6 EAMGIGILTV SEQ ID NO:12 100 0.6 YAAGIGILTV SEQ ID NO:134 15 FAAGIGILTV SEQ ID NO:14 2 30 Influenza A GILGFVFTL SEQ ID NO:17 160 matrix₅₈₋₆₆ MAGE-3₁₆₈₋₁₇₆ EVDPIGHLY SEQ ID NO:20 >100 <0.6

SEQ ID NOS: 1, 5, 6, 9, 10, 13 and 14 all showed higher affinity thanSEQ ID NO:2.

EXAMPLE 4

One concern in developing MHC binding peptides is that the resultingcomplexes of MHC molecule and peptide be stable, preferably more stablethan the peptide originally found complexed to the MHC molecule.

To test stability of the newly synthesized peptides, T2 cells wereincubated, overnight, at room temperature in serum free medium withsaturating amounts of peptide (10 uM), and 3 μg/ml β-microglobulin, tofacilitate the assembly of the requisite MHC molecules. Peptides werethen removed, and 10⁻⁴M ementine (which inhibits protein synthesis) wasadded. The cells were then incubated at 37° C. for varying periods oftime. Aliquots of cells were stained, at various points of theincubation, with a labelled HLA-A2 specific mAb to measure HLA-A2expression.

Stability was determined by comparison with complexes involving SEQ IDNO:17 which were stable over a 6 hour period. The results are presentedin FIGS. 2A-D. FIG. 2A shows show mean fluorescence intensity for eachpeptide. “NC” refers to HLA-A*0201, presented by T2 cells, in theabsence of exogenous peptide, while “FLUMA” is SEQ ID NO:17, and is anacronym for “Flu matrix antigen.” In FIG. 2A, the peptides are SEQ IDNOS: 2, 1 and 9. In FIG. 2B, they are SEQ ID NOS:2, 7, 8, 5, 6 and 17(“FLUMA”). In FIG. 2C, they are SEQ ID NOS: 1, 12, 11, 9, 10 and 17. InFIG. 2D, they are SEQ ID NOS:1 13, 14 and 17. The breakup is solely tofacilitate the review. FIGS. 2B-2D show relative complex stability wherefluorescent intensities with test peptides were normalized, relative tothe stability observed when using SEQ ID NO:17. SEQ ID NOS:1 and 2 bothform unstable complexes, which decay within one hour. This was alsofound with SEQ ID NOS:7 and 8. On the other hand, SEQ ID NOS: 9, 10, 13and 14 formed stable complexes over a 6 hour period, while SEQ ID NOS:5,6, 11 and 12 formed complexes of intermediate stability.

EXAMPLE 5

The antigenic activity of each of the peptides presented, supra, whenthe peptide was associated with HLA-A*0201, was tested in a ⁵¹Cr assayof the type discussed, supra, using TILs, and CTLs. Dose responseanalyses were performed on each peptide, and antigenic activity,relative to SEQ ID NO:2, was calculated. These values are set forth inthe following Tables III and IV and FIGS. 3 and 4 which present datafrom TILs, (Table III and FIG. 3) and CTLs (Table IV and FIG. 4),respectively.

Substituting Ala with Leu or Met enhanced activity between 7.5 and 20fold, while substitutions at the second position nearly abolished it,even though binding to HLA-A*-0201 was increased (Table III and FIG. 3).

SEQ ID NO:1 was better recognized than SEQ ID NO:2, and substitution ofAla in the second position of SEQ ID NO:1 increased recognition 30- and600 fold, respectively. Such substitutions at position 3 reducedactivity, which was expected. Substitution of position 1 resulted in anincrease in recognition.

TABLE III TILN LAU 203 TILN LAU 132 Relative Relative Peptide Sequence[nM] activity [nM] activity AAGIGILTV SEQ ID NO: 2 60 1 30 1 ALGIGILTVSEQ ID NO: 5 >1000 <0.6 >1000 <0.03 AMGIGILTV SEQ ID NO: 6 >1000<0.6 >1000 <0.03 LAGIGILTV SEQ ID NO: 7 6 10 1.5 20 MAGIGILTV SEQ ID NO:8 8 7.5 2.5 12 EAAGIGILTV SEQ ID NO: 1 12 5 3 10 ELAGIGILTV SEQ ID NO: 92 30 0.05 600 EMAGIGILTV SEQ ID NO: 10 2 30 0.05 600 EALGIGILTV SEQ IDNO: 11 >1000 <0.06 >1000 <0.03 EAMGIGILTV SEQ ID NO: 12 >1000<0.06 >1000 <0.03 YAAGIGILTV SEQ ID NO: 13 5 20 1 30 FAAGIGILTV SEQ IDNO: 14 1 60 0.05 600

The results obtained with CTLs are presented herein. Specifically, fiveindependent HLA-A*0201 restricted Melan-A specific CTL clones were used,each of which is known to lyse melanoma target cells.

The CTLs recognized SEQ ID NO:2 with varying efficiency. When Leu wasused to substitute Ala at position 1, four of the five clones showedenhanced recognition, while similar substitutions at position 2 resultedin a loss of activity. Three of the five clones recognized SEQ ID NO:1more efficiently than SEQ ID NO:2, but all recognized SEQ ID NO:9 veryefficiently, while recognition of SEQ ID NO:10 resulted in decreasedefficiency of recognition to differing degrees, and SEQ ID NO:11resulted in reduced recognition for four of five. When SEQ ID NO:12 wastested, it was surprising that recognition improved, because TILrecognition decreased. With respect to SEQ ID NOS: 13 and 14, there wasreduced recognition by the CTLs.

It can be gathered from this that SEQ ID NOS: 7 and 9 were betterrecognized, consistently, than the other peptides tested, while otherpeptides were recognized different degrees.

TABLE IV Recognition of peptide analogs by Melan-A specific CTL clonesM77.86 7.10 Recognition by clone M77.80 1.13 Peptide Peptide PeptidePeptide Peptide SEQ [nM] Relative [nM] Relative [nM] Relative [nM]Relative [nM] Relative ID NO: Peptide sequence 50% activity 50% activity50% activity 50% activity 50% activity 2 AAGIGILTV 15 1 50 1 300 1 300 14000 1 5 ALGIGILTV 90 0.16 >1000 <0.015 >1000 <0.03 >1000 <0.03 >10000<0.4 6 AMGIGILTV >1000 <0.015 >1000 <0.015 >1000 <0.03 >1000<0.03 >10000 <0.4 7 LAGIGILTV 0.08 187 1.5 33 150 2 0.03 10000 30 130 8MAGIGILTV 0.6 .25 15 3 200 1.5 0.5 600 80 50 1 EAAGIGILTV 0.15 100 4 120.06 5000 600 0.5 2000 2 9 EALGIGILTV 300 0.05 >1000 <0.015 40 7.5 >1000<0.3 >10000 <0.4 10 EAMGIGILTV 0.5 30 1 50 0.02 15000 5 60 50 80 11ELAGIGILTV 0.015 1000 0.5 100 0.015 20000 0.5 600 20 200 12 EMAGIGLTV550 36 >1000 <0.015 40 7.5 >1000 <0.3 >10000 <0.4 13 YAAGIGILTV 0.0151000 35 1.4 >1000 <0.3 1000 0.3 >10000 <0.4 14 FAAGIGILTV 0.005 3000 77 >1000 <0.3 >1000 <0.3 200 20 Relative antigenic activity of Melan-Aderived peptides was measured as described in the legend to FIG. 4 andtable III.

EXAMPLE 6

Based upon the preceding data, the peptide of SEQ ID NO:9 was used forCTL induction studies.

In accordance with Valmori, et al, supra, peripheral blood lymphocytesfrom HLA-A*0201 positive melanoma patients were purified bycentrifugation, and were enriched for CD3⁺ cells. The enrichedsubpopulation was then selected for CD8⁺ cells. The resultingsubpopulations routinely contained better than 90% CD8⁺ cells, and thesewere used in further experiments.

The purified, CD8⁺ T cells were plated, at 1-2×10⁶ cells/well, togetherwith 2×10⁶ stimulator cells, the preparation of which is discussed,infra. The effector and stimulator cells were combined in a total of 2ml of Iscove's medium which had been supplemented with 10% human serum,L-arginine (0.55 mM), L-asparagine (0.24 mM), and L-glutamine (1.5 mM),together with recombinant human IL-7 (10 ng/ml).

To prepare the stimulator cells, 2×10⁶ autologous PBLs were incubatedfor 2 hours, at 37° C., in serum free medium, with 20 μg/ml of eachpeptide and 3 μg/ml β-microglobulin. The PBLs were then washed,irradiated, (3000 rads), and then adjusted to an appropriate volume,before being added to the CD8⁺ cell populations. On day 7, cells wererestimulated with peptide pulsed, autologous PBLs in complete medium,supplemented with 10 ng/ml of recombinant human IL-7, and 10 U/ml ofrecombinant human IL-2. There were weekly restimulations, using PBLswhich were peptide pulsed and irradiated. CTL activity was tested forthe first time after the second cycle (MC-2).

The results are shown in the following table and in FIG. 4. In FIG. 4,the source of CD8⁺ cells used was LAU203. CTL activity was assayed sevendays after the second (MC-2) restimulation. Results were obtained usingSEQ ID NOS: 1 and 2. These were used to permit comparison to SEQ ID NO:9.

Note that there was barely any activity with the parental peptides insample LAU203, while SEQ ID NO: 9 provoked a strong CTL response. Thisactivity was also cross reactive with SEQ ID NO: 1.

The results in the following table describe experiments using the samepeptides and using PBL from eight different HLA-A2 positive melanomapatents, LAU203, LAU182, LAU145, LAU86, LAU50, LAU148, LAU161 andLAU119.

Additional experiments are depicted in FIG. 3 which show recognition ofvarious Melan-A peptide analogues presented by T2 cells, by TILN LAU203and TILN LAU132. A four hour ⁵¹Cr assay was conducted at a lymphocyte totarget ration of 30:1.

The first panels of FIG. 3 (top and bottom) compare SEQ ID NOS: 2, 7, 8,5 and 6.

The second set of panels (top and bottom) compare SEQ ID NOS: 1, 9, 10,11 and 12.

The third set (top and bottom) compares SEQ ID NOS: 1, 13, 14 and 4.

The most important result obtained herein, however, was the fact thatCTLS, induced with SEQ ID NO: 9 did recognize and lyse cells presentingthe endogenous peptide when SEQ ID NO: 9 was used.

Percentage specific lysis from cultures stimulated with peptide^(a)) SEQID NO: 2 SEQ ID NO: 1 SEQ ID NO:^(c)) Melan-A 27-35 Melan-A 26-35Melan-A 26-35 A27L SEQ ID NO: 5 tested on: Patient T2 + T2 + T2 + codeE/T^(b)) T2 M10 Me260 Me290 T2 M10 Me290 Me260 T2 M10 Me290 M3260 LAU203 100  38  29 7 17  37 41 15  6 32 83 18  81 30 29  11 10  0 17 23 7 126 96 4 75 10 3  6 2 0  9 17 0 0 17 73 1 62 LAU 132 100  9 12 1 0 19 196 3 34 50 6 31 30 3  7 2 0  5 10 1 2 16 32 3 18 10 0  0 5 1  0  0 1 0  523 2  6 LAU 145 100  15  24 4 1 39 40 5 9 29 50 6 30 30 9 12 3 1 15 25 21 10 29 5 19 10 3  6 0 0  4  6 0 0 10 16 3  7 LAU 86 100  36  29 22  544 38 14  10  35 45 24  15 30 17  15 9 5 20 26 6 0 24 23 10   4 10 16  5 2 0 10 10 1 0 14  9 1  0 LAU 50 100  21  26 7 5 15 20 5 5 19 26 6 2030 7 16 4 5  8 13 1 0 10 18 3  8 10 7  7 0 4  0  4 1 0  3 12 0  0 LAU148 100  51  39 13  4 46 45 9 0 34 39 9  4 30 19   8 5 4 20 26 1 2 19 279  3 10 3  6 1 0 14 14 6 0 13 13 1  0 LAU 161 100  24  22 6 1 33 31 3 125 38 4 23 30 3  8 6 1 16 12 3 0 18 23 2 13 10 2  0 5 0  9  7 2 0  5 113  4 LAU 119 100  31  27 5 12  33 31 1 4 18 45 5 45 30 7 13 1 1 17 23 34 13 39 4 25 10 4  0 0 0  9 12 1 0  7 17 2 16 Clone 6 10 7 73 2 73   3 374 0 61   1 0 65 0 51  ^(a))Lytic activity was assayed 7 days after thethird restimulation. ^(b))Lymphocyte to target cell ratio titration wasperformed for every assay. ^(c))Numbers represent the percent specificlysis obtained for each target. Me290 is a Melan-A and HLA-A*0201positive melanoma cell line obtained from patient LAU203. Me260 is aHLA-A*0201 negative melanoma cell line obtained from patient LAU149 Eachnumber represents the geometric mean of duplicate cultures. Bold facetype indicate significant specific CTL. When the difference in specificlysis obtained on T2 cells in presence or in absence of #Melan-A 26-35(1 μM) or Me290 and Me260 is equal or higher than 10%. A patient isconsidered as responder when a significant specific lysis is detected inat least one of the cultures. ^(d))Clone 6 is a Melan-A specific CTLclone derived from the TILN 289.

The foregoing examples, as will be seen, describe the various featuresof the invention. These include peptides which bind to HLA molecules,such as HLA-A2 molecules, exemplified by HLA-A*0201, which may alsoprovoke proliferation of cytolytic T cells, These peptides, as will beseen from the data herein, are nonapeptides or decapeptides. As with allpeptides, the first amino acid is the amino terminus, and the last oneis the carboxy terminus. The peptides of the invention may bedecapeptides, which have a Val moiety at the C, or carboxy terminus.They may have at the amino terminus, Tyr or Phe when the second aminoacid is Ala. In another embodiment, the amino terminus is Glu followedby Ala, Leu or Met in the second and third position, and terminate withVal, wherein if position two is Ala, position three must be Met or Leu,and vice versa. The peptides having the amino acid sequences set forthin any of SEQ ID NOS:5, and 8-14 are exemplary.

Also a part of the invention are isolated cytolytic T cell lines whichare specific for complexes of these peptides and their MHC bindingpartner, i.e., an HLA molecule, such as an HLA-A2 molecule, HLA-A*0201being especially preferred.

The ability of these peptides to bind to HLA molecules makes them usefulas agents for determining presence of HLA-A2 positive cells, such asHLA-A*0201 positive cells, by determining whether or not the peptidesbind to cells in a sample. This “ligand/receptor” type of reaction iswell known in the art, and various methodologies are available fordetermining it.

A further aspect of the invention are so-called “mini genes” which carryinformation necessary to direct synthesis of modified decapeptides viacells into which the mini genes are transfected. Mini genes can bedesigned which encode one or more antigenic peptides, and are thentransferred to host cell genomes via transfection with plasmids, or viacloning into vaccinia or adenoviruses. See, e.g., Zajac, et al., Int. J.Cancer 71: 496 (1997), incorporated by reference.

Also a feature of the invention is the use of these peptides todetermine the presence of cytolytic T cells in a sample. It was shown,supra, that CTLs in a sample will react with peptide/MHC complexes.Hence, if one knows that CTLs are in a sample, HLA-A2 positive cells canbe “lysed” by adding the peptides of the invention to HLA-A2 positivecells, such as HLA-A*0201 positive cells, and then determining, e.g.,radioactive chromium release, TNF production, etc. or any other of themethods by which T cell activity is determined. Similarly, one candetermine whether or not specific tumor infiltrating lymphocytes(“TILs”) are present in a sample, by adding one of the claimed peptideswith HLA-A2 positive cells to a sample, and determining lysis of theHLA-A2 positive cells via, e.g., ⁵¹Cr release, TNF presence and soforth.

Of course, the peptides may also be used to provoke production of CTLs.As was shown, supra, CTL precursors develop into CTLs when confrontedwith appropriate complexes. By causing such a “confrontation” as itwere, one may generate CTLs. This is useful in an in vivo context, aswell as ex vivo, for generating such CTLs.

Other features of the invention will be clear to the skilled artisan,and need not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

What is claimed is:
 1. An isolated peptide which binds to an HLA-A2molecule and consists of ten amino acids, said isolated peptide havingVal at its carboxy terminus, Glu at its amino terminus, Leu or Met atthe second amino acid from the amino terminus, and Ala at the thirdamino acid from the N terminus (SEQ ID NO: 21).
 2. The isolated peptideof claim 1, wherein the second amino acid from the N terminus is Leu. 3.The isolated peptide of claim 1, wherein the second amino acid from theamino terminus is Met.
 4. An isolated peptide which binds to an HLA-A2molecule and consists of ten amino acids, said isolated peptide havingVal at its carboxy terminus, Glu at its amino terminus Ala at the secondamino acid from the amino terminus and Leu of Met at the third aminoacid from the N terminus (SEQ ID NO: 21).
 5. The isolated peptide ofclaim 4, wherein the third amino acid from the N terminus is Leu.
 6. Theisolated peptide of claim 4, wherein the third amino acid from the Nterminus is Met.